The present invention relates to compositions and methods for detecting hyaluronidase activity in situ and, more particularly, to methods of diagnosing cancer.
Hyaluronan (HA), also known as hyaluronate or hyaluronic acid, is a high molecular weight linear glycosaminoglycan, composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine. HA is an important structural component of cartilage, synovial fluid, skin of vertebrates as well as of the vitreous humor of the eye (1). Hence, HA plays an important role in maintenance of intact architecture in normal tissues; it absorbs a large volume of water and creates a gel-like environment. This environment enables facilitating biological processes that require cell movement such as tissue organization and morphogenesis, wound healing, inflammation, and angiogenesis, as well as in pathological processes such as cancer metastasis.
High molecular weight HA is degraded by hyaluronidase (hyal). Low molecular weight degradation products are known to stimulate endothelial cell proliferation and to promote neovascularization, while the native high molecular weight HA is anti-angiogenic (2). Hyaluronidase was first identified in 1928 as a “spreading factor” of viral agents and was later characterized as HA-degrading enzyme and named hyaluronidase (3-5). Hyaluronidase activity is high in necrotic tumors and is elevated in the sera of cancer patients (6). Six hyal-like gene sequences, coding for ubiquitous enzymes with varying substrate specificities, were described in mammals based on their enzymatic activity and optimal pH for activity (7).
Primarily investigated hyaluronidase protein products are Hyal-1, Hyal-2 and PH-20. Hyal-1, the plasma hyaluronidase is a 57-kDa protein composed of a single polypeptide chain of 49 kDa with approximately 8 kDa of post-translational glycosylation. Hyal-1 is a lysosomal enzyme that can cleave HA to small tetra-and-disaccharides (8). Hyal-1 is found at high concentrations in the urine and has approximately 40% identity to the enzyme PH-20 found mainly in sperm (7). Hyal-1 is also known as LUCA-1 (LUng CAncer-1) defined by functional tumor suppressor activity (9). Hyal-2 is anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) link. It cleaves high molecular weight HA to approximately 20 kDa fragments. It seems to function as either an oncogene or a tumor suppressor gene. Over expression of Hyal-2 accelerates tumor formation of murine astrocytoma cells (10), but can also accelerate apoptosis (11). The secretion of hyaluronidase by cancer cells can contribute to their aggressiveness and invasiveness. Thus, hyaluronidase provides the intermediate HA fragments that induce angiogenesis (12). Moreover, secretion of hyaluronidase by the cancer cell enables digestion of the HA barrier and thus facilitate invasion of tumor cells to neighboring organs and tissues. It was reported that hyaluronidase activity in ovarian cancer tissue is significantly higher than in endometrial cancer tissue. A significant correlation was found, between hyaluronidase activity and metastasis of ovarian cancer (13). Moreover, treatment of conditioned media with hyaluronidase increased the adhesion of ovarian cancer tumor cells to mesothelial monolayer which served as a model for metastatic dissemination in the peritoneal cavity (14). Since HA hyaluronidase and CD44 are involved in ovarian carcinoma (15-18), and the presence of hyaluronidase can be a sign for the presence of metastases, it is important to detect hyaluronidase activity in a non-invasive, sensitive and specific way.
To date several methods are being used for the detection of hyaluronidase including quantitative spectroscopic ELISA-like assay using avidin biotin peroxidase complex (19), a microtiter-based assay for hyaluronidase activity (20), the Morgan-Elson reaction (21) and its fluorimetric version (22) and chromatography (23). However, none of these methods can be applied in-situ for non-invasive imaging.
While reducing the present invention to practice, the present inventors designed a novel approach for in-situ detection of hyaluronidase activity, which may be used for diagnosing cancer such as, ovarian cancer.